high throughput analysis of the satellitome illuminates satellite dna evolution

www.nature.com/scientificreports OPEN received: 14 January 2016 accepted: 02 June 2016 Published: 07 July 2016 High-throughput analysis of the satellitome illuminates satellite DNA evolution Francisco J. Ruiz-Ruano, María Dolores López-Ln, Josefa Cabrero & Juan Pedro M. Camacho Satellite DNA (satDNA) is a major component yet the great unknown of eukaryote genomes and clearly underrepresented in genome sequencing projects Here we show the high-throughput analysis of satellite DNA content in the migratory locust by means of the bioinformatic analysis of Illumina reads with the RepeatExplorer and RepeatMasker programs This unveiled 62 satDNA families and we propose the term “satellitome” for the whole collection of different satDNA families in a genome The finding that satDNAs were present in many contigs of the migratory locust draft genome indicates that they show many genomic locations invisible by fluorescent in situ hybridization (FISH) The cytological pattern of five satellites showing common descent (belonging to the SF3 superfamily) suggests that non-clustered satDNAs can become into clustered through local amplification at any of the many genomic loci resulting from previous dissemination of short satDNA arrays The fact that all kinds of satDNA (micro- mini- and satellites) can show the non-clustered and clustered states suggests that all these elements are mostly similar, except for repeat length Finally, the presence of VNTRs in bacteria, showing similar properties to non-clustered satDNAs in eukaryotes, suggests that this kind of tandem repeats show common properties in all living beings Eukaryote genomes are plenty of repetitive elements including transposable elements (TEs), tandem repeats, segmental duplications, ribosomal DNA, multi-copy gene families, pseudogenes, etc which, collectively, constitute the repeatome1 Satellite DNA consists of a single sequence tandemly repeated many times, in contrast to tandemly repeated genes (e.g ribosomal RNA and histone genes) where the repeating unit consists of several different DNA sequences (i.e genes and spacers) Satellite DNA has been classified into microsatellites, minisatellites and satellites, with no complete consensus about the precise length limits2,3 Although satellite DNA has traditionally been considered to be junk DNA, some possible functions have been suggested during last years One of the most accepted functional roles for satDNA is its implication in centromeric function4, but other possible functional roles have also been suggested in relation with heterochromatin formation through the siRNA pathway5,6 The name “satellite DNA” is historical since this kind of repetitive DNA was discovered as a small peak in the CsCl ultracentrifugation profile7 Today this technique is not performed to search for satDNA, since it was replaced by other techniques such as DNA renaturation kinetics8, restriction digestion and electrophoresis yielding a ladder pattern9 and, most recently, by the bioinformatic analysis of a huge collection of short DNA sequences yielded by Next Generation Sequencing (NGS)10 Anyway, the term “satellite DNA” is still useful because it is simple, descriptive and profusely used in the literature On this basis, we are proposing here the name “satellitome” for the whole collection of satDNAs in a genome The recent publication of a draft genome of the migratory locust (Locusta migratoria) represents a milestone as it is the largest animal genome hitherto sequenced11 There is no doubt that it has provided excellent information for performing genomic work in other insects even though annotation is not complete However, as in other sequenced genomes, information about the repetitive components of the genome is rather scarce, especially for satDNA We have recently reported microsatellite content in L migratoria at both genomic and cytogenetic levels12, but the search for satDNAs through the classical restriction endonuclease digestion and electrophoresis approach failed in this species (MD López-León and P Lorite, personal communication) Up to now, only 21 satDNAs have been reported in 12 orthopteran species, most of them grasshoppers (Supplementary Table S1) Recently, the use of NGS and new bioinformatic tools like RepeatExplorer10 has allowed the high-throughput detection of repetitive DNA, including satellite DNA, the most extreme case being the plant Luzula elegans with Departamento de Genética, Facultad de Ciencias, Universidad de Granada, Granada, Spain Correspondence and requests for materials should be addressed to J.P.M.C (email: jpmcamac@ugr.es) Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ 37 satDNA families, 20 of which were analyzed by FISH13 Here we perform the high-throughput analysis of the satellitome from the information contained in Illumina reads obtained from two individuals of the migratory locust representing the Southern (SL) and Northern (NL) lineages (see methods) By means of stepwise clustering of repetitive DNA10 intermingled by substraction of the repetitive elements found in previous steps, we found that the satellitome of L migratoria consists of, at least, 62 different satDNAs with monomer size ranging between and 400 bp This procedure allowed detection of many poorly abundant satDNAs which would have gone unnoticed through conventional methods The physical mapping of 59 of them by FISH showed three types of chromosome distribution, with clear predominance of chromosome-specific satDNAs Finally, this broad catalog of different satDNA families allowed an analysis for general features which provided new insights on the origin and evolution of this part of the repeatome Results High-troughput search for satDNAs.  In the first run of RepeatExplorer (RE), performed in parallel on Illumina reads from the SL and NL individuals, we found 26 and 21 satDNAs, respectively We then selected all available reads of each lineage which, after DeconSeq filtering, lacked homology with all satDNAs and other RE clustered sequences previously found in both lineages A new RE run detected 11 new satDNAs in SL and in NL After a new step of filtering and RE analysis, we found new satDNAs in SL and in NL However, the next filtering and RE step failed to show any new satDNA in both lineages, for which reason we stopped this iterative process At the end, we found 39 satDNAs in the SL individual and 35 in the NL individual As a whole, these analyses revealed the existence of 62 different satDNA families, 27 of which were assembled in SL, 23 in NL and 12 in both lineages Subsequent analyses with RepeatMasker to score satDNA abundance and divergence, revealed that 59 out of the 62 satDNAs were present in both lineages, whereas two of them (LmiSat31-8 and LmiSat43–231) were not found in the NL individual, and one (LmiSat62-23) was not found in the SL individual (Table 1) The analysis of variation within these 62 satDNA families showed the presence of 107 sequence variants (i.e 1–7 per family) (Table 1, Supplementary Table S2, Supplementary Fig S1) Collectively, all 62 satDNAs represent about 2.39% of the Southern genome and 2.74% of the Northern one (Table 1 and Supplementary Fig S2) This low amount of satDNA is consistent with the low amount of constitutive heterochromatin revealed by C-banding in this species14 The high number of different satDNA families found in the genome of the migratory locust and the plant L elegans13 indicates that eukaryote genomes usually contain a high diversity of satDNA families During next years, huge amounts of new satDNAs are expected to be uncovered using NGS approaches We therefore suggest the following simple nomenclature rules to help managing this new information: satDNA name should begin with species abbreviation in Repbase (e.g Lmi for Locusta migratoria) followed by the term “Sat”, a catalog number in order of decreasing abundance (according to the first genome analyzed), followed by consensus monomer length For instance, the most abundant satDNA in the Spanish genome of L migratoria would read LmiSat01–193 The catalog number would allow differentiating two satDNAs coinciding in length If, in the future, additional satDNA families were found in other populations of the same species, they should be numbered subsequently to the last one described in previous work Optionally, if a function is assigned to a satDNA, a reference to it could be added at the end of the name For instance, since we know that LmiSat07-5 in L migratoria is the telomeric DNA repeat15, we could name it LmiSat07-5-tel SatDNA abundance was very similar in both genomes, but divergence showed a tendency to be higher in the Northern genome (Supplementary Results S1) To test the reliability of the satDNAs found, we designed primers in opposite orientation for all of them and PCR amplified 59 of them on genomic DNA from Spanish specimens, belonging to the Southern lineage, collected at Cádiz The three exceptions (LmiSat46–353, LmiSat52–143 and LmiSat57–230) were rare satDNAs which had been found by RepeatExplorer only in the NL genome, whereas RepeatMasker detected them also in the SL individual from the Padul population However, PCR failed to amplify them in four different SL individuals from the Cádiz population, suggesting population differences for the presence of these rare satDNAs In addition, LmiSat07-5-tel corresponded with the telomeric DNA repeat (TTAGG)15, and was excluded from subsequent analyses because of its known function Therefore, we will work here with the remaining 58 satDNAs The 58 satDNAs showed high variation for monomer length (8–400 bp) and A +​ T content (29.4–67.6%) (Table 1) Monomer length showed a bimodal distribution, with a 37 bp gap (between 90 and 127 bp) dividing the 58 satDNAs into two groups, one including 26 short satDNAs (8–90 bp) and the other comprising 32 long satDNAs (127–400 bp) The 37 bp gap in monomer length appears to be an oddity of the L migratoria genome, as we have not found such a long gap in L elegans or other grasshopper species (Ruiz-Ruano et al., unpublished) Long satDNAs showed higher A +​ T content and lower divergence than short ones, and the latter show a very high tendency to arise from G +​ C-rich genomic regions (Supplementary Results S2) Short and long satDNAs show similar patterns of chromosomal location.  We performed single FISH analysis for all 58 satDNAs and also double FISH combining a satDNA probe with rDNA or histone gene probes, when needed for accurate identification of the satDNA-carrying chromosomes Both short (Fig. 1) and long (Fig. 2) satDNAs showed three main patterns at cytological level: clustered at specific chromosome regions (c), non-clustered (nc) and a mixed pattern (m) (Table 1) Depending on satDNA abundance, the non-clustered pattern can go from complete absence of FISH signal to general chromosome brightness above background The mixed pattern includes both large and very small clusters The frequencies of c, nc and m patterns did not differ significantly between the two length classes (Supplementary Results S3) As a whole, the 47 clustered satDNAs (excluding telomeric DNA) showed 89 chromosomal clusters per haploid genome, i.e 1.89 per satDNA and 7.42 per chromosome pair, on average Most of them were proximal Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ L migratoria genome (NL)11 Abundance Divergence V SL NL SL NL 59.59 0.98225 0.6903 4.67 5.07 332 15 176 53.41 0.47509 0.9996 5.32 5.38 12931 100 LmiSat03–195 195 58.97 0.29481 0.2305 5.42 5.96 1003 206 LmiSat04–18 18 50 0.06194 0.0816 7.2 7.23 108 156 i,d i c LmiSat05–400 400 51.25 0.05431 0.0483 4.65 5.04 91 i,d p c LmiSat06–185 185 59.46 0.0541 0.07 4.76 5.28 274 42 LmiSat07–5-tel 60 0.04438 0.1611 1.75 6.12 57 2868 SF SatDNA Family Length A + T LmiSat01–193 193 LmiSat02–176 Contigs MNRPC LmiSat08–168 168 57.74 0.03737 0.0467 4.96 4.91 327 28 LmiSat09–181 181 60.22 0.02944 0.0072 5.38 7.42 45 60 LmiSat10–9 55.56 0.02269 0.029 11.79 11.42 267 243 LmiSat11–37 37 62.16 0.01873 0.0069 7.75 8.12 317 106 LmiSat12–273 273 56.41 0.01836 0.0113 3.5 5.29 23 16 LmiSat13–259 259 57.53 0.01697 0.0115 4.38 6.25 137 27 LmiSat14–216 216 51.85 0.01426 0.0091 5.39 8.79 70 40 LmiSat15–190 190 55.26 0.01426 0.0166 4.09 4.5 212 LmiSat16–278 278 62.59 0.0139 0.0082 2.49 3.01 17 LmiSat17–75 75 57.33 0.01177 0.0033 5.79 6.66 112 LmiSat18–210 210 60.48 0.01121 0.0267 6.33 4.59 LmiSat19–89 89 60.67 0.01058 0.0034 3.82 6.44 10 LmiSat20–15 15 53.33 0.01032 0.0201 12.71 14.15 190 256 LmiSat21–38 38 50 0.01013 0.0019 2.85 20 LmiSat22–17 17 58.82 0.01 0.0092 10.81 10.28 182 426 LmiSat23–223 223 61.43 0.00927 0.0106 4.42 5.73 18 10 3 2.91 LmiSat24–266 266 56.39 0.00895 0.0066 2.06 5.14 51 LmiSat25–219 219 39.73 0.00834 0.0105 5.88 8.2 21 LmiSat26–240 240 66.2 0.00809 0.00436 7.44 9.25 33 LmiSat27–57 57 47.37 0.0079 0.0103 8.99 9.66 333 326 LmiSat28–263 263 57.41 0.00768 0.0139 1.79 2.22 91 12 LmiSat29–68 68 58.82 0.00719 0.0019 9.36 14.48 46 89 LmiSat30–138 138 40.58 0.0068 0.0055 5.74 9.03 23 83 37 12 LmiSat31–8 50 0.00668 LmiSat32–261 261 51.72 0.00631 0.0056 5.98 3.86 9.18 LmiSat33–21 21 47.62 0.00627 0.0039 7.77 8.35 30 179 LmiSat34–299 299 61.87 0.00622 0.0048 6.81 7.39 406 LmiSat35–228 228 55.7 0.00597 0.0053 2.43 4.64 25 18 L2 X M3 M4 M5 M6 M7 M8 S9 p p p p p p p p p p p p c p p p c p p p p p t p S10 S11 Pattern t c p t t t t t t p p t t t p c t c p m p c p p p c c d c p c i,i i p c c d c p c p c p c nc i,d m i c i d c d nc d c i c nc i,i c p c p c p p c d c i c p c nc LmiSat36–15 15 60 0.00585 0.0093 16.88 15.12 279 302 LmiSat37–238 238 66 0.00544 0.00224 6.53 6.52 111 37 LmiSat38–42 42 64.29 0.00511 0.0046 14.56 14.94 106 692 LmiSat39–53 53 32.08 0.00503 0.0013 6.79 9.17 14 119 i c LmiSat40–148 148 67.57 0.00459 0.0023 2.35 3.05 20 d c LmiSat41–180 180 61.67 0.00455 0.0058 3.38 2.14 LmiSat42–127 127 51.18 0.00447 0.0012 2.02 4.6 2 LmiSat43–231 231 53.68 0.0044 44 LmiSat44–17 17 29.41 0.00428 0.0005 11.45 11.3 53 LmiSat45–274 274 54.01 0.0042 0.0066 8.2 7.22 152 12 LmiSat46–353 353 59.77 0.00407 0.0071 15.49 11.38 1799 12.46 13.22 0.68 LmiSat47–41 41 41.46 0.00369 0.0058 LmiSat48–220 220 58.18 0.00366 0.0011 3.8 48 394 7.74 18 LmiSat49–47 47 42.55 0.00362 0.0113 6.24 6.7 127 282 LmiSat50–16 16 56.25 0.00331 0.0169 8.31 8.24 54 64 LmiSat51–241 241 63.9 0.00294 0.0058 7.32 3.97 33 138 LmiSat52–143 143 51.75 0.00257 0.0076 22.15 14.01 1796 3 Chromosome location (SL) L1 LmiSat53–47 47 40.43 0.00248 0.019 3.16 5.2 23 LmiSat54–272 272 56.25 0.00244 0.0051 4.55 4.15 164 51 LmiSat55–90 90 35.56 0.00164 0.0074 15.62 8.57 nc i i c c i m i p c i c p p,i p p c c p c nc p c i i c c i p i i p d c m nc Continued Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ SF L migratoria genome (NL)11 Abundance Divergence V SL NL SL NL 52.63 0.00083 0.0067 5.09 4.31 15 97 63.04 0.00052 0.0047 18.21 3.4 212 25 SatDNA Family Length A + T LmiSat56–19 19 LmiSat57–230 230 Contigs MNRPC Chromosome location (SL) L1 L2 X M3 M4 M5 M6 M7 M8 p S9 S10 S11 Pattern i m LmiSat58–86 86 41.86 0.00008 0.0127 5.99 3.12 10 nc LmiSat59–16 16 43.75 0.00004 0.0049 18.23 14.54 13 13 nc LmiSat60–255 255 52.94 0.00004 0.0053 1.03 0.99 0 nc LmiSat61–63 63 42.86 0.00002 0.0062 14.99 4.6 11 LmiSat62–23 23 43.48 4.57 Total 0.0045 107 2.39241 2.7417 Total p nc p 3 c 52 Total i 3 0 1 10 26 Total d 0 0 1 0 11 Total loci 12 11 3 7 19 10 89 satDNAs 11 10 3 7 16 10 83 Table 1.  Length (nt), A + T content (%), number of variants (V), abundance (% of the genome), divergence (%), number of contigs found in the draft genome of Locusta migratoria11, maximum number of repeats per contig (MNRPC), chromosome location (in the Southern lineage) and clustering pattern of all 62 satDNA families and superfamilies (SF) In each family, length and A +​ T content are given for the most abundant variant Divergence per family is expressed as percentage of Kimura divergence Chromosome location was analyzed by FISH in a Spanish population SL =​ Southern lineage, NL =​  Northern lineage Chromosome locations: t =​  telomeric, p  =​ proximal to centromere, i =​  interstitial, d  =​  distal Chromosome distribution patterns: c =​  clustered, nc  =​  non-clustered, m  =​ mixed When a satDNA showed two loci in a same chromosome, their locations were indicated separated by a comma Totals at the bottom not include LmiSat07–5 (the telomeric repeat) (52), whereas only 26 were interstitial and 11 distal, with a similar distribution between short and long satDNAs (Supplementary Results S3) With the exception of the telomeric repeat, short satDNAs were clustered on only or chromosome pairs, whereas clustered long satDNAs were found on 1, 2, 3, 5, or all 12 chromosome pairs, the latter condition being found only for LmiSat01–193, which was located proximal to the centromeric region in all chromosomes, with clusters in the eight shortest chromosome pairs (M4-S11) being larger than those in the four longer chromosomes (L1, L2, X and M3) (Fig. 2g) The most frequent pattern, in both short and long satDNAs, was the presence of a large cluster in a single chromosome pair, as was the case for 15 short and 18 long satDNAs (see Supplementary Table S3 and some examples in Figs 1b,d,e and 2e,f), with LmiSat21–38 and LmiSat28–263 showing two clusters in the same chromosome One satDNA (LmiSat23–223) showed the same location as 45S rDNA in this species The ideogram in Fig. 3 summarizes the location of all satDNAs Excluding the two only satDNAs which were present in all chromosomes, i.e LmiSat01–193 and LmiSat075-tel, the remaining 46 families (19 short and 27 long) of clustered satDNAs (including those showing the mixed pattern) were irregularly distributed among the different chromosomes, with four chromosomes lacking short satDNAs (L1, X, M5 and M8) but all chromosomes carrying one or more different long satDNAs, in addition to LmiSat01–193 (Table 1) Remarkably, the S9 chromosome was the only chromosome carrying more short (10) than long (6) satDNAs Only 14 satDNAs (4 short and 10 long) showed clusters in more than one chromosome pair, and this allows testing the equilocality of satDNA distribution As Supplementary Table S4 shows, short and long satDNAs displayed similar equilocality indices (0.63 and 0.65, respectively) thus reinforcing their similarities in chromosome distribution pattern The high number of different satDNAs described here is very useful for chromosome identification in L migratoria, as 15 short and 18 long satDNAs were chromosome-specific markers allowing the direct identification of out of the 12 chromosome pairs, the only exceptions being L1, M6 and S10 (Fig. 3 and Supplementary Table S3) However, these three chromosome pairs can indirectly be identified through their satDNA content pattern, since L1 is the only L-chromosome carrying LmiSat03–195, LmiSat37–238 and LmiSat45–274, M6 is the only M-chromosome carrying LmiSat56-19 and 45S rDNA, and S10 can be identified because it lacks the chromosome-specific satDNAs present in the two similar-sized autosomes (S9 and S11) (e.g LmiSat04–18, LmiSat05–400 and LmiSat06–185) A search for the 62 satDNA sequences in the draft genome of L migratoria11 revealed that most of them were present in a surprisingly high number of contigs, with very high differences among satDNA families (Table 1), this variation being positively correlated with abundance (Spearman rank correlation: rs =​  0.46, N  =​  58, P =​ 0.00026) Remarkably, clustered satDNAs showed no significant difference in the number of contigs compared with non-clustered ones (Mann-Whitney test: U =​  198, P  =​ 0.23), suggesting that both types of satDNAs are similarly scattered throughout the genome Therefore, in addition to the large arrays present in the clusters revealed by FISH, clustered satDNAs show many short arrays at many loci across the genome Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ Figure 1.  Physical mapping of seven of the short satDNAs found in Locusta migratoria, showing the three patterns of chromosome distribution observed: non-clustered (a), clustered (d–g) and mixed (b,c) (a–c) show haploid mitotic metaphase cells from haplo-diploid embryos, whereas (d–g) show diploid cells from normal embryos Each cell is shown in red color for satDNA FISH (upper panel) and merged with DAPI (lower panel) In (e,f) double FISH was performed to distinguish whether the sat-carrying chromosome was L2 instead of L1 (e) and whether S9 carried LmiS at04–18 in addition to rDNA (shown in green color) (f) Inset in (f) shows the S9 chromosome stained with DAPI, on the left, and submitted to double FISH for LmiSat04–18 (red) and rDNA (green), on the right, which was selected from another cell showing lower chromosome condensation Note the presence of three about similar sized satDNA blocks located in interstitial and distal regions of the S9 chromosome In (g) note that LmiSat07–5-tel shows the typical pattern of telomeric repeats Homologies between satDNAs define five superfamilies.  A comparison of DNA sequence between the 58 monomer families revealed the existence of similarity between some of them, which allowed defining five superfamilies (Table 1) As shown in Supplementary Fig S3, superfamily (SF1) includes two long satDNA families: LmiSat01–193, located in pericentromeric regions of all chromosomes, and LmiSat13–259 located only in the M4 chromosome, thus being a case of local derivation of LmiSat13–259 from LmiSat01–193 SF2 includes LmiSat12–273 and LmiSat16–278 both distally located on the L2 chromosome, thus showing satDNA divergence without movement to non-homologous chromosomes SF3 is composed of five different long satDNA families showing all patterns of chromosome location, thus illustrating how long satDNAs may evolve through sequence diversification and changes in chromosome location patterns (Table 1 and Fig. 4) SF4 includes three long satDNA families (LmiSat26–240, LmiSat37–238 and LmiSat51–241) interstitially located on different chromosomes (S11, Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ Figure 2.  Physical mapping of eight of the long satDNAs found in Locusta migratoria, showing the three patterns of chromosome distribution observed: non-clustered (a), clustered (d–h) and mixed (b–c) All cells showed here (except that in (g)) are mitotic metaphase haploid cells from haplo-diploid embryos obtained in our laboratory The cell in (g) is at meiotic metaphase I and was obtained from an adult male Each cell is shown in red color for satDNA FISH (upper panel) and merged with DAPI (lower panel) In (f,g), double FISH was performed to distinguish whether the sat-carrying chromosome was M6 (harboring rDNA shown in green) or any other medium-sized chromosome Note in (h) the presence of LmiSat01–193 in the pericentromeric regions of all chromosomes L1 and L2, respectively), thus providing evidence for clustering on different non-homologous chromosomes Finally, SF5 included three short satDNAs (LmiSat31-8, LmiSat50-16 and LmiSat59-16) showing different location patterns, but the reliability of this superfamily is doubtful (see Supplementary Fig S4 and Supplementary Table S5) Homology with other repeated sequences.  We found seven satDNA families with homology to sequences from Orthoptera contained in Repbase (Supplementary Table S6 and Supplementary Fig S5) LmiSat06–185 showed homology with a satDNA previously described in the grasshopper Caledia captiva16, whereas the six remaining matches in Repbase were with transposable elements (TEs) LmiSat02–176 showed homology with the 5′​-end of a Helitron lineage Two long satDNAs (LmiSat15–190 and LmiSat34–299) showed homology with the CDS of TEs type Gypsy and Polinton, respectively Likewise, LmiSat29–68 andLmiSat55–90 aligned with a region outside the CDS of two different hAT transposons, and LmiSat19–89 with a DNA transposon described in L migratoria In addition, LmiSat07–5-tel is the telomeric DNA repeat conserved in the majority of insects15 Finally, LmiSat11–37 showed high variation for the number of repeats of a GA microsatellite, for which reason this satDNA showed the highest divergence (56%) and number of variants (7) No other satDNA Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ Figure 3.  Ideogram showing chromosome location of satDNA clusters mapped by FISH SatDNAs are noted here only by the catalog number, which is underlined in the case of chromosome-specific families Polymorphic loci are indicated by an asterisk Pericentromeric light-grey areas represent constitutive heterochromatin The inset on the left shows a histogram of monomer lengths for the 62 satDNA families Note the gap between 90 and 127 bp Figure 4.  Minimum spanning tree for SF3 superfamily The link size between haplotypes is proportional to the number of substitutions (s) and indels (id) In brackets, it is indicated the sum of nucleotides involved in the indels SF3 was composed of six sequences corresponding to five different satDNAs, with lengths ranging between 231 and 274 bp Note that they constitute a heterogeneous collection of satDNAs showing common descent and displaying all patterns of chromosome location and thus illustrating how long satDNAs may evolve by changing sequence and chromosome location patterns (see Table 1) carrying microsatellites was found Taken together, these results suggest the possibility that some satDNAs in L migratoria originated from TEs, as in other organisms17,18 Discussion The 62 satDNA families of L migratoria reported here constitute the highest number of satDNA families ever found in a non-model species The closest case was the 37 satDNAs reported in the plant Luzula elegans within a normal run of RepeatExplorer yielding 291 major repeat clusters with genome proportions of at least 0.01%13 Remarkably, the application of our filtering approach to the Illumina reads deposited by Heckman et al.13 in SRA uncovered 85 satDNA families (grouped into superfamilies), with genome proportions of 0.00035% or higher (Supplementary Table S7) This indicates that our approach improves significantly the bioinformatic analysis for satDNA characterization with RepeatExplorer, by being able to find satDNAs showing 28-fold lower abundance By performing several successive filtering steps and searches with RepeatExplorer, in each step subtracting those repetitive elements found in previous steps, the chance of finding other poorly represented satDNAs is substantially increased In L migratoria, the use of genomic reads from two distant populations has also been very useful, allowing detection of satDNAs with abundance as low as 0.00002% Anyway, it is still conceivable the existence of Scientific Reports | 6:28333 | DOI: 10.1038/srep28333 www.nature.com/scientificreports/ other less abundant satDNA families which have gone unnoticed with our methodology Likewise, other individuals from the same or a different population could harbour other satDNA variants or families The high-throughput analysis of the satellitome in L migratoria has unveiled several interesting properties of this kind of tandem repeats: (1) The “library” hypothesis19 predicts that related species share an ancestral set of different conserved satellite DNA families which may be differentially amplified in each species due to stochastic mechanisms of concerted evolution20 The Northern and Southern lineages of L migratoria have shown very similar satellitome catalogs, with only slight differences indicating differential amplification between individuals and/or populations The intraspecific library shown by the L migratoria satellitome is not composed of completely independent satDNAs, as some of them show similarities enough to constitute five superfamilies Remarkable conservation was displayed by LmiSat06–185, which showed 72.2% similarity with a satDNA described in Caledia captiva (Acridinae subfamily)16, a species sharing the most recent common ancestor with L migratoria (Oedipodinae subfamily) about 47 million years ago21 SatDNA conservatism has been reported in several organisms, such as beetles genus Palorus22, the human alpha-satellite DNA (which is highly conserved in chicken and zebrafish)23 and satDNAs in some plants24, the most extreme case being the persistence of a satDNA for 540 million years in bivalve mollusks25 The satellitome opens new avenues to test the library hypothesis at several phylogenetic levels, and library catalogs will be known in unsuspected detail thanks to the NGS techniques (2) Short and long satDNAs showed the same three patterns of chromosome location (non-clustered, clustered or mixed), and similar equilocal distribution across non-homologous chromosomes In consistency with previous observations on minisatellites26, the short satDNAs observed in L migratoria tend to show high G +​ C content and sequence divergence, the latter being especially apparent when they are interspersed into euchromatin (3) The observed equilocality for short and long clustered satDNAs indicates that heterochromatin equilocality27 (i.e the tendency to occupy similar location on non-homologous chromosomes) is actually based on satDNA equilocality, and this pattern may be facilitated by telomere reunion at first meiotic prophase bouquet28 which, in the case of acrocentric chromosomes, also implies the reunion of centromeres Remarkably, short and long satDNAs showed very similar tendency to equilocality (4) Satellite DNA is frequently located into heterochromatin, and this feature is used to define this kind of DNA In L migratoria, constitutive heterochromatin is restricted to small pericentromeric regions14, which thus include the 52 pericentromeric clusters found for 26 satDNAs However, the 26 interstitial (for 21 satDNAs) and 11 distal (for 10 satDNAs) clusters are outside constitutive heterochromatin in this species Therefore, we conclude that satellite DNA is also contained into euchromatic regions, in consistency with recent findings in Drosophila29 and Tribolium castaneum30 (5) The high-throughput analysis of the satellitome has been highly informative on satellite DNA evolution Our present results suggest that previously defined types of satellite DNA3 (microsatellites, minisatellites and satellites) show similarities at genomic and cytological levels We have found here satDNAs with monomer length reaching the domains of typical microsatellites, such as the 5 bp telomeric repeat in L migratoria or several satDNAs in L elegans showing monomer lengths of only or 6 bp (Supplementary Table S7) Likewise, about half of the satDNAs found in L migratoria showed monomer lengths like those defining minisatellites (​ 2 0, using the options “ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN: [100/101]” We randomly selected 2 ×​ 250,000 reads with SeqTK (https://github.com/lh3/seqtk) and run RepeatExplorer with default options and a custom database of repeated sequences, in addition to Repbase v20.1044, last accessed October 28, 2015 We manually selected the clusters with spherical or ring-shaped structure and density values (i e the mean number of links per read) being higher than 0.1 For each cluster we chose the contigs showing the highest coverage and generated a dotplot with Geneious v4.845 If we detected tandem structure, we split the contigs in monomers to align them and generate a consensus monomer for each contig We then chose a new collection of reads and those that matched previously detected satDNAs were filtered out with the DeconSeq v0.4.3 software46, with default options, before a new RepeatExplorer run was performed We used satDNA dimers as reference and, in case of dimers shorter than 200 bp, we concatenated so many monomers as needed to surpass this length The mismatched reads were then assembled in a new run of RepeatExplorer to search for the presence of satDNAs being poorly represented in the crude reads but detectable in the filtered ones This procedure increased very much the number of analyzed reads without dramatically increasing computational effort Therefore, we run RepeatExplorer with 2 ×​ 500,000 filtered reads, searched for new satDNAs and filter them out We repeated this process two more times adding 2 ×​ 500,000 reads in each iteration, until no new satDNA was detected by RepeatExplorer We mined satDNAs following the same steps in parallel for the gDNA libraries from the Northern and Southern lineages For satDNA sequence analysis, we compared the consensus sequences of all satDNAs found in order to investigate possible homology between some of them For this purpose, we aligned each satDNA against the whole satDNA catalog with RepeatMasker v4.0.547, using the Cross_match search engine, recording all matches between satDNAs When sequences showed less than 80% of identity we considered them as different satDNA families sharing a same superfamily Sequences showing identity higher than 80% were considered variants of the same family, and those showing identity higher than 95% were considered the same variant We numbered satDNA families in order of decreasing abundance in the Southern lineage individual [GenBank:KU056702–KU056808] We built a minimum spanning tree for DNA sequences in each superfamily with Arlequin v3.548, considering each indel position as a single change and representing the relative abundance among Southern and Northern individuals We used RepeatMasker47 with “-a” option to estimate abundance and divergence for each satDNA variant in gDNA libraries We selected 2 ×​ 5 millions of paired reads where all nucleotides met quality criteria applied for the satMiner protocol Abundance estimates provided by RepeatMasker showed highly significant positive correlation with those yielded by RepeatExplorer in both the Southern (Spearman rs =​  0.84, N  =​  15, P  =​  0.000074) and Northern (rs =​  0.97, N  =​  17, P